Chronic Rhinosinusitis (CRS) is one of the most frequently reported chronic diseases in the U.S. Even with aggressive medical and surgical therapies, many CRS patients have persistent disease. The pathogenesis of CRS is not well understood, and the etiology, classification, and assessment of disease severity remain controversial. Human airways are constantly exposed to environmental fungal spores, often at higher levels than the levels of allergenic pollens. During the past funding period, peripheral blood immune cells from CRS patients reacted to these common fungi, and a product(s) of an environmental fungus, Alternaria, showed a unique immunostimulatory activity. These findings led to a novel hypothesis;namely, CRS is caused or exacerbated by an exaggerated immune response to airborne fungi, such as Alternaria. Airway immune cells in CRS patients produce vigorous immune and inflammatory responses to fungi and fungal products, especially to protease(s) and a glycolytic enzyme(s), resulting in uncontrolled Th1 and Th2 cytokine production, persistent eosinophilic inflammation, tissue damage and remodeling. To test this hypothesis, the immune responses in CRS patients to common airborne fungi will be characterized by in vitro and in vivo approaches (Aim 1). Immune responses from both innate and acquired immune cells in blood and sinus tissues of CRS patients will be compared to the responses in allergic and nonallergic controls. The product(s) of Alternaria, which triggers profound Th2-like inflammation in vitro in human airway cells and in vivo in murine airways, will be identified and characterized (Aim 2). After proteomic identification, the recombinant molecule(s) and gene knockout fungus will be examined for their biological activities in human airway and immune cells in vitro and in mice in vivo. This application involves a multidisciplinary team of immunologists, allergy physicians, otorhinolaryngology surgeons, and functional genomic scientists. Elucidation of the mechanisms of persistent airway inflammation in CRS will advance the field and help to provide specific and effective therapies for this common, costly, and incapacitating disorder. Relevance: This proposal will investigate why patients with chronic rhinosinusitis have such persistent health problems. It will use advanced biotechnological methods to examine common environmental agents that may cause or intensify this disease.